Myb transcriptional repressor asmyb054 related to synthesis of albicanone and application thereof

Abstract

This invention discloses a MYB transcriptional repressor, AsMYB054, related to the synthesis of 2-(2-phenylethyl) chromones in Aquilaria sinensis and its applications. Targeting the weak links in the synthesis and regulation of 2-(2-phenylethyl) chromone compounds, this invention clones for the first time the AsMYB054 gene, a transcriptional repressor related to the regulation of 2-(2-phenylethyl) chromone synthesis in Aquilaria sinensis. AsMYB054 is located in the cell nucleus and possesses transcriptional repressive activity, capable of binding to and inhibiting the activity of the promoters of enzyme genes AsPKS02 and AsPKS09, which are related to the biosynthesis of 2-(2-phenylethyl) chromone compounds. Inhibiting the expression of AsMYB054 using gene editing or RNA interference techniques can promote the accumulation of 2-(2-phenylethyl) chromone compounds in Aquilaria sinensis, showing broad application prospects and significant economic value.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, specifically to a MYB transcriptional repressor AsMYB054 related to the synthesis of sclerotinia scabra chromone and its applications. Background Technology

[0002] Agarwood is a traditional and precious medicinal material and natural fragrance, formed from the resinous wood of plants in the genus *Aquilaria* (family Thymelaeaceae) after injury. Due to its high value and large international market demand, *Aquilaria* plants have long been subject to over-logging. *Aquilaria sinensis* (white agarwood) is the only source plant of agarwood listed in the *Chinese Pharmacopoeia* (2020 edition) and is a national second-class endangered protected plant. Under natural conditions, agarwood formation is extremely slow, requiring decades or even centuries of accumulation. Therefore, methods for artificially inducing agarwood formation have been developed, including mechanical damage induction, chemical induction, fungal induction, and whole-tree resin formation. Artificial induction methods have accelerated agarwood formation to some extent, but the formation mechanism is still not fully understood, and there is still significant room for improvement in the quality and yield of artificially induced agarwood. 2-(2-phenylethyl)chromone compounds are the main characteristic components of agarwood and key components for identifying authenticity and evaluating quality; their relative content is an important indicator for judging agarwood quality. The formation of 2-(2-phenylethyl)chromone compounds is thought to be a result of environmental stress. Polyketide synthases (PKS) are key enzymes in their biosynthesis, but the regulatory mechanism of their biosynthesis is still unclear.

[0003] Transcription factors can bind to the promoters of genes involved in the biosynthesis of secondary metabolites, activating or inhibiting gene expression to participate in the accumulation of secondary metabolites, thereby improving plant adaptability to the environment. MYB transcription factors are one of the largest family of plant transcription factors, and their members play important roles in various physiological processes such as secondary metabolism and responses to biotic and abiotic stresses. However, MYB transcription factors related to the biosynthesis of 2-(2-phenylethyl)chromone compounds in Aquilaria sinensis have not yet been reported. Researching the regulation of MYB transcription factors on the biosynthesis of 2-(2-phenylethyl)chromone compounds will enhance our understanding of the biosynthesis and regulation of characteristic components of Aquilaria sinensis, and is of great significance for developing novel and efficient resin-inducing technologies and improving the yield and quality of Aquilaria sinensis through gene editing or transgenic technologies. Summary of the Invention

[0004] Therefore, embodiments of the present invention provide a MYB transcriptional repressor AsMYB054 related to the synthesis of sclerotinia sclerotinia and its application.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of the present invention, the present invention provides a gene of AsMYB054, a MYB transcriptional repressor related to the synthesis of 2-(2-phenylethyl)chromone compounds from Aquilaria sinensis, the gene having the following sequence:

[0007] (a) The nucleotide sequence shown in SEQ ID NO:1;

[0008] (b) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1, wherein one or more nucleotides have been replaced, deleted, and / or added, and which expresses a protein with the same function; or,

[0009] (c) A nucleotide sequence that has at least 90% homology with the nucleotide sequence of (a) or (b) and expresses the same functional protein.

[0010] According to a second aspect of the present invention, the present invention provides a MYB transcriptional repressor AsMYB054, which is related to the synthesis of 2-(2-phenylethyl)chromone compounds from Aquilaria sinensis, having the following sequence:

[0011] (a) The amino acid sequence shown in SEQ ID NO:2; or,

[0012] (b) The amino acid sequence shown in SEQ ID NO:2 is replaced, deleted or added with one or more amino acids to form an amino acid sequence with equivalent function.

[0013] According to a third aspect of the present invention, the present invention provides a recombinant vector comprising the MYB transcriptional repressor AsMYB054 gene as described above.

[0014] According to a fourth aspect of the present invention, the present invention provides the application of the MYB transcriptional repressor AsMYB054 gene, as described above, in the early identification of high-quality agarwood seedlings. Specifically, high-quality agarwood seedlings are identified by detecting the expression level of said gene.

[0015] According to a fifth aspect of the present invention, the present invention provides the application of the MYB transcriptional repressor AsMYB054 gene as described above in artificial agarwood inoculants, transgenic plants, and gene-edited plants. Specifically, the gene is applied to artificial agarwood inoculants, transgenic plants, and gene-edited plants to improve the yield and quality of agarwood.

[0016] This invention discloses AsMYB054, a MYB transcriptional repressor related to the biosynthesis of 2-(2-phenylethyl) chromone compounds in Aquilaria sinensis, and its applications. Using transcriptome data from Aquilaria sinensis treatment, AsMYB054, a MYB transcriptional repressor involved in the biosynthesis of 2-(2-phenylethyl) chromone compounds, was screened and cloned. Its coding region is 816 bp in length, encoding 271 amino acids. AsMYB054 contains conserved C1 and C2 transcriptional repression domains and belongs to the S4 class of MYB transcriptional repressors. Subcellular localization and transcriptional activity analysis showed that AsMYB054 is located in the cell nucleus and possesses transcriptional repressive activity. AsMYB054 can bind to and inhibit the promoters of AsPKS02 and AsPKS09, indicating that AsMYB054 can inhibit the expression of the AsPKS02 or AsPKS09 genes, thereby participating in the regulation of the biosynthesis of 2-(2-phenylethyl) chromone compounds. Inhibiting the expression of AsMYB054 using gene editing or RNA interference techniques can promote the accumulation of 2-(2-phenylethyl)chromone compounds in Aquilaria sinensis. This invention enhances our understanding of the biosynthesis and regulation of characteristic components of aquilaria sinensis and is of great significance for developing novel and efficient resin-inducing technologies and improving the yield and quality of aquilaria sinensis through gene editing or transgenic technologies.

[0017] The embodiments of the present invention have the following advantages:

[0018] 2-(2-phenylethyl)chromones are the main characteristic components of agarwood, and their relative content is an important indicator for judging the quality of agarwood. This invention addresses the weak links in the synthesis and regulation of 2-(2-phenylethyl)chromones by cloning, for the first time, the transcriptional repressor gene AsMYB054, which is related to the regulation of 2-(2-phenylethyl)chromone synthesis in Aquilaria sinensis. AsMYB054 is located in the cell nucleus and possesses transcriptional repressive activity, capable of binding to and inhibiting the activity of the promoters of enzyme genes AsPKS02 and AsPKS09 related to the biosynthesis of 2-(2-phenylethyl)chromones. Inhibiting the expression of AsMYB054 using gene editing or RNA interference technology can promote the accumulation of 2-(2-phenylethyl)chromones in Aquilaria sinensis, showing broad application prospects and significant economic value. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] Figure 1Phylogenetic analysis of AsMYB054 and other species' MYB transcriptional repressors with the Arabidopsis MYB transcription factor family;

[0021] Figure 2 Molecular characterization analysis of AsMYB054. A. Sequence alignment and conserved domain analysis of AsMYB054 with S4 class MYB transcription factors from other species; B. Transcriptional activity analysis of AsMYB054; C. Subcellular localization analysis of AsMYB054.

[0022] Figure 3 To investigate the regulation of polyketide synthase gene expression by AsMYB054. A) Yeast one-hybrid assay to identify the interaction between AsMYB054 and the PKS gene promoter; B) EMSA assay to identify the interaction between AsMYB054 and the AsPKS02 and AsPKS09 promoters; C) Dual luciferase reporter gene system to detect the effect of AsMYB054 on the activity of the AsPKS02 and AsPKS09 promoters. Detailed Implementation

[0023] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Cloning of the MYB transcriptional repressor AsMYB054 from Aucklandia lappa

[0025] By analyzing the transcriptome sequencing results of Aquilaria sinensis stems treated with a resin-forming agent in the early stage, specific primers were designed: P1 forward primer 5′-ATGGGTAGATCTCCTTGCTGTG-3′ (SEQ ID NO:3) and P2 reverse primer 5′-TCATTTCATCTCCAAACCTCTAT-3′ (SEQ ID NO:4). Using the first-strand cDNA of Aquilaria sinensis stems as a template, the CDS sequence of the AsMYB054 gene was amplified by PCR under the following conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 70℃ extension for 1 min, for a total of 35 cycles; 70℃ extension for 5 min. The PCR product was ligated into the pMD18-T vector, transformed into E. coli competent cells, and positive clones were screened and sequenced to obtain the full-length coding sequence of the AsMYB054 gene, 816 bp. The recombinant vector containing the AsMYB054 gene was named pMD18-AsMYB054.

[0026] Example 2: Sequence characteristics of the MYB transcriptional repressor AsMYB054 from Aucklandia lappa

[0027] The full-length open reading frame (ORF) of the AsMYB054 gene from *Saussurea involucrata* is 816 bp (SEQ ID NO:1). Based on the ORF sequence, the amino acid sequence of AsMYB054 was deduced, consisting of 271 amino acid residues (SEQ ID NO:2), with a molecular weight of 30.59 kDa and a theoretical isoelectric point of 8.75.

[0028] Using MEGA7 software, AsMYB054 was compared with Arabidopsis MYB transcription factors and other MYB transcription repressors in plants using Clustalw. The comparison results were analyzed using the NJ method, and a phylogenetic tree was constructed using Bootstrap with a selection of 1000. The analysis showed that AsMYB054 and MYB transcription repressors belong to the S4 subfamily (…). Figure 1 This family of proteins has been shown to be negative regulators of the biosynthesis of secondary metabolites such as phenylpropanoids, betaines, and tanshinone. DNMAN sequence alignment results show that AsMYB054 and other S4 subfamily proteins have similar structural features, containing conserved C1 and C2 transcriptional repressor domains. Figure 2 A). Therefore, it is speculated that AsMYB054 belongs to the S4 class of MYB transcriptional repressors and may have similar functions.

[0029] Example 3: Transcriptional activity analysis of AsMYB054

[0030] To verify whether AsMYB054 possesses transcriptional activity, AsMYB054 was ligated into the PGBKT7 and PGBKT7-V16 vectors (which possess transcriptional activation activity). The empty PGBKT7 vector served as a negative control, and PGBKT7-V16 as a positive control. Results showed that both the control and experimental groups grew normally on a single-deficient (SD / -Trp) medium, indicating successful plasmid transformation into yeast. However, on a triple-deficient medium containing X-α-gal (SD / -Trp-His-Ade-X-α-gal), only the positive control grew normally and showed a blue color; the others did not grow. Figure 2 B). The results showed that AsMYB054 is a transcription factor with transcriptional repression function.

[0031] Example 4 Subcellular localization of AsMYB054 protein

[0032] Driven by the constitutive promoter CaMV35S, the recombinant vector AsMYB054-GFP and the control empty vector pNC-Cam1304-SubC were transformed into onion epidermal cells. Fluorescence microscopy revealed that the control vector exhibited green fluorescence throughout the cells, while the recombinant vector AsMYB054-GFP showed a strong green fluorescence signal only in the cell nucleus. Figure 2 C). This indicates that AsMYB054 is located in the cell nucleus and functions as a transcriptional regulator there.

[0033] Example 5 Interaction between AsMYB054 and the promoter of the PKS gene, an enzyme related to the synthesis of 2-(2-phenylethyl)chromone compounds

[0034] Polyketide synthases (PKS) are key enzymes in the biosynthesis of 2-(2-phenylethyl)chromone compounds in agarwood. Transcriptome analysis of stems from *Aquilaria sinensis* treated with resin-forming agents revealed that seven PKS genes may be involved in the biosynthesis of 2-(2-phenylethyl)chromone compounds. This study aimed to verify whether AsMYB054 binds to the promoter of PKS genes, a key enzyme in the synthesis of 2-(2-phenylethyl)chromone compounds. We first cloned the promoters of the AsPKS01 (1390 bp), AsPKS02 (1467 bp), AsPKS03 (1711 bp), AsPKS06 (1590 bp), AsPKS07 (1351 bp), AsPKS08 (1261 bp), and AsPKS09 (1272 bp) genes, and ligated them into the bait vector His2.1. The recombinant bait plasmid was named AsPKSpro-His2.1. Simultaneously, AsMYB054 was ligated into the pGADT7 prey vector, and the recombinant prey plasmid was named AsMYB054-AD. The constructed bait plasmid and prey plasmid were co-transformed into Y187 competent yeast cells and plated on two-deficient medium (SD / -Trp-Leu) and three-deficient medium (SD / Leu / Trp / -His) containing different concentrations of 3-AT, respectively. The results showed that yeast cells grew well only when AsMYB054-AD was co-transformed with AsPKS02pro-His2.1 or AsPKS09pro-His2.1 in the presence of 3-AT, while yeast cells did not grow when AsPKS02pro-His2.1 or AsPKS09pro-His2.1 was co-transformed with the empty vector pGADT7. Figure 3 A). The results show that AsMYB054-AD can interact with the AsPKS02 and AsPKS09 promoters and directly target and bind to the AsPKS02 and AsPKS09 genes.

[0035] To further verify the interaction between AsMYB054 and the AsPKS02 and AsPKS09 promoters, an electrophoretic migration and retention assay (EMSA) was performed. First, the AsMYB054 coding region was ligated into the prokaryotic expression vector pNC-ET28a via seamless cloning, transformed into E. coli BL21 competent cells, and induced with IPTG. The AsMYB054-His tag protein was purified using HiTrapFF Crude. The 1467 bp AsPKS02 and 1272 bp AsPKS09 promoters were cloned and purified using a DNA purification kit (Foregene, DE-03011). EMSA experiments were performed using an EMSA kit (Invitrogen, E33075). The purified AsMYB054 was incubated with double-stranded promoter nucleotides at room temperature for 30 minutes. The DNA / protein complex sample was then subjected to 12% polyacrylamide gel electrophoresis at 120V for 30 minutes. Finally, the gel was stained with SYBR Green EMSA for analysis. The results showed that AsMYB054 could bind to the AsPKS02 or AsPKS09 promoter and induce motility transfer; the amount of migrating DNA gradually increased with increasing protein concentration. Figure 3 B). The results further confirm that AsMYB054 can interact with the promoters of AsPKS02 and AsPKS09, and the strength of the interaction depends on the concentration of AsMYB054.

[0036] Example 6: Regulation of the PKS gene for the synthesis of 2-(2-phenylethyl)chromone compounds by AsMYB054

[0037] Using a dual-luciferase reporter gene detection system, this study investigated whether AsMYB054 regulates the activity of the AsPKS02 or AsPKS09 promoters. AsMYB054 was ligated into the pGreenII62SK vector to generate the effect plasmid AsMYB05-pGreenII62SK. The AsPKS02 or AsPKS09 promoters were ligated into pGreenII 0800-LUC to generate the reporter plasmids AsPKS09pro-pGreenII 0800-LUC and AsPKS02-pGreenII 0800-LUC, respectively. The effect plasmids and reporter plasmids were transformed into Agrobacterium GV3101, and the cultured Agrobacterium cells were collected by centrifugation and suspended in MES+MgCl2 suspension. The suspensions containing the effect vectors were then mixed with the suspensions containing the reporter vectors at a 2:1 ratio. The suspensions were then injected into tobacco leaves. Three days later, the REN / LUC ratios of the two luciferases in the leaves were measured to analyze the regulatory effect of AsMYB054 on the promoter activity of the AsPKS02 and AsPKS09 genes. The results showed that the REN / LUC ratios of AsPKS02pro-pGreenll 0800-LUC+AsMYB05-pGreenII62SK and AsPKS09pro-pGreenll 0800-LUC+AsMYB05-pGreenII62SK were 41% and 78% lower than the control, respectively. Figure 3 C) indicates that AsMYB054 can inhibit the expression of the AsPKS02 or AsPKS09 genes, thereby participating in the regulation of the biosynthesis of 2-(2-phenylethyl)chromone compounds.

[0038] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A gene of MYB transcription inhibitor AsMYB054 related to synthesis of 2-(2-phenylethyl) chromone compounds in Aquilaria sinensis, characterized in that, The sequence of the gene is: the nucleotide sequence shown in SEQ ID NO:

1. 2.A MYB transcription inhibitor AsMYB054 related to synthesis of 2-(2-phenylethyl) chromone compounds in A. sinensis characterized in that, The sequence is: the amino acid sequence shown in SEQ ID NO:

2.

3. A plant comprising the MYB transcriptional repressor of claim 1 AsMYB054 recombinant vectors of the genes.

Images